A portable lateral flow distance-based paper sensor for drinking water hardness test

Hardness is one of the basic parameters of water, and a high-level hardness of drinking water may be harmful to human health. Thus, it is very important to monitor drinking water hardness. In this work, a portable lateral flow distance-based paper sensor for the semi-quantitative detection of drinking water hardness is demonstrated. In the presence of Ca2+/Mg2+, the hydrogel can be formed via the chelation between sodium alginate and Ca2+/Mg2+, inducing a phase separation process. The viscosity change of the sodium alginate solution is directly related to the Ca2+/Mg2+ concentration and can be determined by the water lateral flow distance on test strips. The sensor successfully realizes the quantification of Ca2+ and Mg2+ in the range of 0–10 mmol L-1 and 4–20 mmol L-1, respectively. The recoveries are found varied from 95% to 108.9%. The water hardness is acceptable for drinking if the Cr values lies in the range of 0.259 to 0.419, and it is high with the Cr value above 0.595. Remarkably, the performance of the sensor is comparable with the commercial kit for real water samples, which avoids the subjective judgment. Overall, this method provides a portable approach for semi-quantitative detection of drinking water hardness with the merits of convenience and low cost, which shows great potential for the potential application.


Introduction
Water is an indispensable natural resource for human beings.Drinking water quality is vital to human health.Water hardness is one of the basic parameters for evaluating water quality, and a high-level drinking water hardness may cause diseases [1,2].Water hardness is generally referred to the sum content of calcium, magnesium, iron, aluminum, zinc and other ions contained in water, and usually calculated by Ca 2+ and Mg 2+ contents considering the lower concentrations of other ions, which is also called as Ca 2+ hardness.Although the Ca 2+ concentration in drinking water is not strictly defined, the World Health Organization recommends that calcium ion level in drinking water should be no more than 5 mmol L -1 [3,4].Therefore, it is very important to detect Ca 2+ hardness in drinking water samples.
Until now, several methods for monitoring Ca 2+ have been reported despite its difficulty to be differentiated from other interfering ions.Among them, atomic absorption spectrometry and complexometric titration are the classic methods used for calcium quantification [5,6].However, they often suffer from the disadvantages such as complex procedures, bulky sample requirement, complicated instrumentation, and trained operators.For instance, the commercial colorimetric kit has already been accessible, which is developed based on the titration method.However, a large amount sample of 10 mL was generally required.Meanwhile, the color change of titration terminal is subjectively judged by operators, which may inevitably lead to systematic errors.Additionally, the fluorescence method is available for the simplified detection process with high sensitivity [7][8][9], but it still requires the usage of large-scale instrument and trained operators.Ionophore-based ion-selective optode also have been used for the colorimetric detection of Ca 2+ [10,11].Nevertheless, the results are greatly influenced by the pH of the samples, which hampers their broad application.Thus, it is greatly demanded to construct a portable sensor for monitoring Ca 2+ in water.
Paper-based detection methods have become appealing in recent years, with the merits of convenient operation, fast response, and easy modification.They are extensively applied in clinical diagnostics, food quality management, and environmental monitoring [12][13][14][15].In particular, the distance-based paper sensor can quantify the analyte by measuring the change of water flow distance, which has shown great application prospect as point-of-care test, because of its advantages of simple portability, visualization, easy quantification, and short analysis time [16][17][18].The pH indicator papers can be employed as test strips because they are cheap and can clearly show the water flow marks.In addition, analyte-responsive hydrogels are series of polymers with three dimensional network structures.Various hydrogels have been designed and developed for biosensing of metal ions [19,20], nucleic acids, proteins [21], and microorganisms [22,23].With the specific experimental design, hydrogels can respond to analytes and induce physical or chemical changes that generate subsequent readable signals.Among these methods, the gel-sol transition triggered by external stimuli is a most commonly used detection principle [23][24][25].Therefore, it possesses great potential for the exploration of the distance-based lateral flow sensor using stimuli-responsive polymers for the evaluation of drinking water hardness.
Herein, a portable lateral flow sensor with the distance readout signal for the semi-quantitative determination of drinking water hardness on the paper strip was developed (Scheme 1).In the presence of Ca 2+ /Mg 2+ , the sodium alginate (Alg) hydrogel network with "egg-box" structure can be formed during the phase separation process.Correspondingly, the viscosity of the Alg solution drops sharply due to reduction of the Alg concentration.Thus, the concentration of Ca 2+ /Mg 2+ can be determined via the measurement of water lateral flow distance on paper strips of the residual Alg solution.This method offers a simple and convenient method for the water Ca 2+ hardness evaluation using a small amount of samples with satisfacoty accuracy, which also avoids the subjective color endpoint judgment.

Optimization of the sodium alginate concentration
Tris-HCl buffer (100 mmol L -1 , pH = 7.4) was used in this study.The Alg solutions with different concentrations from 0.1 wt% to 0.5 wt% were firstly prepared at 25˚C.The solutions of CaCl 2 with various concentrations of 0, 2, 4, 10, 20 mmol L -1 were then obtained.According to our previous study, the mixture of CaCl 2 and Alg solution was incubated at 25˚C after vortex for 30s [26].Then, 30 μL of the supernatant solution obtained by centrifuging for 1 min was transferred onto the left side of the test strip.After waiting for 2 min, the images of the paper sensor were captured by smartphone and analyzed by Adobe Photoshop software.All of the experiments were conducted at least three times to obtain the standard deviations.

Determination of the water hardness using the commercial test kit
Firstly, 10 mL of the test solution was accurately pipetted into the cleaned conical flask, followed by the addition of a package of total hardness reagent I.If the solution color is pure blue after reagent I dissolved, the hardness value of the water sample is 0 mg/L.If the solution exhibits purple red, hardness reagent II aqueous solution was then vertically added with the continuous shaking of the conical flask.The dropping speed should be controlled as 1 drop every 3 s until the solution changed from purple red to pure blue.Then, the number of drops consumed (N) was recorded and hardness (mg L -1 , calculated as CaCO 3 , 1 mg L -1 = 0.01 mmol L -1 ) was calculated by the following equation, the detection range of this kit is 30-600 mg L -1 :

Data analysis
The pH indicator papers were photographed with a smartphone.Then, the pixel values of areas including water marked and whole test strips were obtained by Adobe Photoshop and recorded as P mark and P total , respectively.Finally, the ratio of P mark and P total was calculated as the water trace coverage ratio (Cr) as follows: and Alg were 10 mmol L -1 and 0.2 wt%, respectively.Moreover, the similar variation tendency was observed with different concentrations of Alg (Fig 2C -2F).The results are reasonable considering the crosslinking process.At the initial stage, the addition of Ca 2+ into Alg solution can cause the rapid formation of the hydrogel based on the chelation between them, leading to the increased viscosity.While the continued increase of the Ca 2+ concentration contributed to increase of the cross-linking degree of the hydrogel, which was separated from the aqueous phase.As only the remainly Alg was in the aqueous phase, the viscosity is decreased with further increase of the Ca 2+ concentration.Thus, the viscosity change can be coupled to the concentration change of Ca 2+ .As concluded from

Detection of Ca 2+ and Mg 2+
Inspired by the excellent performance of the paper sensor above, the study investigating determination of the concentration of Ca 2+ and Mg 2+ was also conducted.Based on the results in Fig 2, the Cr value droped at the Ca 2+ concentration from 0-4 mmol L -1 and increased at the Ca 2+ concentration from 4-20 mmol L -1 , which makes it challenging for the direct quantitative detection of Ca 2+ .Thus, 4 mmol L -1 Ca 2+ was initially added directly into the test sample to

The selectivity of the paper sensor
The Ca 2+ of 10 mmol L -1 was chosen in this section.The water flow distance remained almost same with different pH values from 5-9 (Fig 4A ), which indicated the pH stability of the paper sensor.Subsequently, the influence of ionic strength of the solution was also considered (Fig 4B).The actual drinking water samples rarely contains heavy metal ions.Therefore, common anions were selected for selectivity detection.The results show that ignorable difference was observed at a fixed Ca 2+ concentration of 10 mmol L -1 in the presence of NaCl solution (0-200 mmol L -1 ).The corresponding photograph is shown in S4 Fig.

The semi-quantitative detection of water hardness using the paper sensor
The World Health Organization recommends that the Ca 2+ level in drinkable water should be less than 5 mmol L -1 .Thus, the corresponding Cr value of drinkable water sample detected by the paper sensor should be in the range of 0.259 to 0.595 in the absence of Mg 2+ according to Fig 3B .If only Mg 2+ is existing in the tested sample, the concentration of Mg 2+ should be below than 12.5 mmol L -1 Mg 2+ for drinkable water, which means the same water hardness and calculated by the equation: According to Fig 5, the Cr values should be below 0.419.In short, the water hardness is suitable for drinking if the Cr values lie in the range of 0.259 to 0.419, and unqualified with Cr value above 0.595.Thus, the proposed sensor has been successfully applied for the semi-quantitative detection of water hardness.

Comparison of the paper sensor with the commercial kit
The applicability of the paper sensor was also evaluated in water.The standard addition method was used herein.After 4 mmol L -1 of Ca 2+ was initially added into the tested purified water, the additional different concentrations of Ca 2+ with 0, 2, 4 and 6 mmol L -1 were spiked The recoveries were found varied from 95% to 108.9% and from 105% to 145% for the paperbased sensor and the commercial kit, respectively (Table 1).The results also exhibited the comparable accuracy of the proposed paper sensor with the commercial kit.Test-t analysis was performed between the proposed paper-based method and the ICP-MS method which is the gold standard for metal content measurement.The p values were calculated as 0.317, 0.353, and 0.596 respectively, corresponding to the marked samples with Ca 2+ concentrations of 2, 4, 6 mM.No significant difference were observed, which demonstrated the reliability of the paper method.More importantly, only 30 μL of the test sample is required for the paper sensor, which is much less than the amount of the test sample (10 mL) used for the commercial kit.In addition, the comparison of this paper-based sensor with different methods for the detection of Ca 2+ and Mg 2+ were performed (S1 and S2 Tables), which clearly demonstrated the merits of low-cost, convince, and easily-read for the paper-based sensor.Thus, this method works as an effective means for the quantification of Ca 2+ in water.

Conclusions
In summary, this work reported a portable distance-based lateral flow paper-based sensor for drinking water hardness semi-quantitative detection based on the phase separation induced by  the gel-sol transition.Briefly, the chelation between sodium alginate and Ca 2+ /Mg 2+ triggers the formation of hydrogel to cause the viscosity changes of sodium alginate solution.The viscosity change can be quantified by the water flow distance on test strips.This method effectively avoids the usage of a large amount of samples, complicated operation, and high cost, which also exhibits high accuracy performance.Though the paper sensor could not exclude thoroughly the interference produced by other metal ions in some specific water samples, this approach can eliminate the error caused by the subjective color endpoint judgment in tested commercial kit, which shows the great potential of the paper sensor for further application.

Fig 2 ,
the fixed 0.2 wt% Alg solution was chosen for following experiments, because it can clearly distinguish different concentrations of Ca 2+ .

Fig 2 .
Fig 2. Optimization of conditions for the paper sensor.(A) The Cr values of the paper sensor responses towards the Alg solutions with different concentrations.(B)-(F) The responses of the paper sensor towards different concentrations of Alg with different concentrations of Ca 2+ (0, 2, 4, 10 and 20 mmol L -1 ).https://doi.org/10.1371/journal.pone.0308424.g003 Finally, the potential interfering ions in real samples including NaCl, KCl, NaH 2 PO 4 , NaH 2 PO 4 , (NH 4 ) 2 S 2 O 8 , Na 2 S 2 O 3 , NaHSO 3 and NaHCO 3 were mixed with Ca 2+ .As shown in Fig 4C and 4D, the water flow distances exhibited no obvious alternation.All of these results suggest the satisfactory selectivity of the paper sensor.

Fig 3 .
Fig 3. Detection of Ca 2+ and Mg 2+ .(A) The images of the sensors and (B) the linear plot between the Cr and Ca 2+ concentration from 4-14 mmol L -1 .(C) The images of the sensors and (D) the linear plot between the Cr and Ca 2+ concentration from 0-10 mmol L -1 .(E) The images of the sensors and (F) the linear plot between the Cr and Mg 2+ concentration from 4-20 mmol L -1 .https://doi.org/10.1371/journal.pone.0308424.g004

Fig 4 .
Fig 4. The selectivity of the paper sensor.(A) The Cr values responses for the mixture solution of Alg and Ca 2+ with different pH values, (B) for aqueous solutions with NaCl solutions (0 to 200 mmol L -1 ) and Ca 2+ , (C) for aqueous solutions with other ions and Ca 2+ , and (D) the corresponding photohraphs, respectively.The concentration of each ion was 10 mmol L -1 .https://doi.org/10.1371/journal.pone.0308424.g005

Fig 5 .
Fig 5.The illustration of the semi-quantitative detection of water hardness using the paper sensor.(A) The Cr values and (B) corresponding images of the paper sensor under different conditions: in the presence of 4 mmol L -1 Ca 2+ + 12.5 mmol L -1 Mg 2+ , 4 mmol L -1 Ca 2+ + 5 mmol L -1 Ca 2+ , and 4mmol L -1 Ca 2+ .(C) The Cr values ranges illustration of the sensor for the water hardness detection for the drinking water.https://doi.org/10.1371/journal.pone.0308424.g006